This proposal focuses on molecular mechanisms of signal transduction by G-protein coupled receptors (GPCR). Mammalian rhodopsin is the prototypic receptor in this family and is the major focus of the work herein proposed. A unifying hypothesis is proposed that in all GPCR, the molecular mechanisms of signal transduction (after light activation in rhodopsin and after ligand binding in GPCR in general) are conserved and similar. In rhodopsin, the first consequence of light-induced retinal isomerization has been established to be movements of helices in the transmembrane domain. These movements induce a specific conformational change in the cytoplasmic face. The tertiary structure changes involved have previously been studied by this PI by biochemical (chemical reactivity, accessibility and proximity relationships between amino acids) and by spin labeling and EPR spectroscopy. These will now be studied more precisely by NMR. Both, solution NMR (19F and TROSY,) and solid state NMR will be used. Large scale expressions of proteins which are necessary for NMR experiments will be carried out by further investigation of HEK293 stable cell lines and by the Drosophila expression system. Protein-protein interactions leading to sensitization and desensitization are at the heart of signal transduction by rhodopsin. Detailed kinetic studies of interactions between light-activated rhodopsin, transducin and rhodopsin kinase will be carried out by the techniques of surface plasmon resonance using a Biacore instrument. A further focus in studies of protein-protein interactions is to define the interacting sites and amino acid sequences by covalent cross-linking methods. The interacting regions will be identified by mass spectrometric methods. Finally, it is now feasible to test the unifying hypothesis proposed for signal transduction by studying helix movements on ligand binding in two members of GPCR, alpha1 adrenergic receptor and beta2 adrenergic receptor.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028289-22
Application #
6476578
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Chin, Jean
Project Start
1980-12-01
Project End
2003-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
22
Fiscal Year
2002
Total Cost
$742,143
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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Kim, Jong-Myoung; Hwa, John; Garriga, Pere et al. (2005) Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops. Biochemistry 44:2284-92
Eilers, Markus; Ying, Weiwen; Reeves, Philip J et al. (2002) Magic angle spinning nuclear magnetic resonance of isotopically labeled rhodopsin. Methods Enzymol 343:212-22
Klein-Seetharaman, J; Reeves, P J; Loewen, M C et al. (2002) Solution NMR spectroscopy of [alpha -15N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence. Proc Natl Acad Sci U S A 99:3452-7
Reeves, Philip J; Kim, Jong-Myoung; Khorana, H Gobind (2002) Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc Natl Acad Sci U S A 99:13413-8
Niu, Li; Kim, Jong-Myoung; Khorana, H Gobind (2002) Structure and function in rhodopsin: asymmetric reconstitution of rhodopsin in liposomes. Proc Natl Acad Sci U S A 99:13409-12
Reeves, Philip J; Callewaert, Nico; Contreras, Roland et al. (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc Natl Acad Sci U S A 99:13419-24
Cai, K; Itoh, Y; Khorana, H G (2001) Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent. Proc Natl Acad Sci U S A 98:4877-82
Kim, J M; Booth, P J; Allen, S J et al. (2001) Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin. J Mol Biol 308:409-22

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